The invention relates to the production of fatty acids and fatty acid products in transgenic or genetically modified organisms, such as microorganisms and photosynthetic organisms. The invention also relates to genes encoding enzymes that function in the biosynthesis of fatty acids and related products, and in particular to acyl-ACP thioesterases.
Plants supply most of the oils used in food products, and plant-derived lipids are also used in the manufacture of many non-dietary products, such as lubricants, soaps, detergents, cosmetics, and thickeners. In higher plants, fatty acids are synthesized in plastids and incorporated into triacylglycerols (triglycerides) in the endoplasmic reticulum (ER). In cells that store fats, such as the cells of seeds or nuts, fat droplets bud off of the ER to form lipid bodies within the cytoplasm. These reservoirs of lipids in the form of triglycerides provide an energy resource for germinating seeds.
The diversity of fatty acids produced by plant cells and incorporated into triglycerides can add to the time and cost of purification of fatty acids of particular chain lengths used for particular purposes. Medium chain fatty acids, for example, are used in the manufacture of surface disinfectants, anti-foaming agents, surfactants, lubricants, perfumes, dyes, and flavoring agents, and can be used to produce polymers and fuels. Long chain fatty acids are used in food products as well as detergents, soaps, surfactants, cosmetics, plastics, and lubricants, and can also be used in the production of fuels.
Acyl-acyl carrier protein (ACP) thioesterases are key enzymes in determining the chain lengths of fatty acids produced by a plant. Two families of acyl-ACP thioesterases are present in higher plants, the “Class I” acyl-ACP thioesterases encoded by FatA genes, and responsible for cleaving long chain (for example, C16 and C18) unsaturated fatty acids from acyl-ACP and the “Class II” acyl-ACP thioesterases encoded by FatB genes, that are active on saturated fatty acyl chains and can be specific for medium chain (C8-C14) acyl-ACPs or can be active on both medium and long chain fatty acyl-ACPs. Different acyl-ACP thioesterases have different degrees of chain length specificity, sometimes referred to as the enzyme's “preference” for cleaving a particular length of fatty acid from ACP, and thioesterases are typically most active in cleaving a particular chain length fatty acid while having lesser activity in cleaving one or more other chain length fatty acids. Some Class II (FatB) acyl-ACP thioesterases have binary activity, having a first peak of activity against a specific medium chain length acyl substrate and a second peak of activity against one or more specific long chain length acyl substrates.
The isolation of Class II acyl-ACP thioesterase genes from higher plants with medium chain specificity or having activity on both medium and long chain fatty acids has been described previously. Examples include U.S. Pat. No. 5,298,421, entitled “Plant medium-chain-preferring acyl-ACP thioesterases and related methods”, which describes the isolation of an acyl-ACP thioesterase and the gene that encodes it from the immature seeds of Umbellularia californica. Other patents of interest include U.S. Pat. No. 5,304,481, entitled “Plant thioesterase having preferential hydrolase activity toward C12 acyl-ACP substrate”, U.S. Pat. No. 5,344,771, entitled “Plant thioesterases”, U.S. Pat. No. 5,455,167, entitled “Medium-chain thioesterases in plants”, U.S. Pat. No. 5,512,482, entitled “Plant thioesterases”, U.S. Pat. No. 5,639,790, entitled “Plant medium-chain thioesterases”, U.S. Pat. No. 5,667,997, entitled “C8 and C10 medium-chain thioesterases in plants”, U.S. Pat. No. 5,807,893, entitled “Plant thioesterases and use for modification of fatty acid composition in plant seed oils”, U.S. Pat. No. 5,850,022, entitled “Production of myristate in plant cells”, and U.S. Pat. No. 5,910,631, entitled “Middle chain-specific thioesterase genes from Cuphea lanceolata”, U.S. Pat. No. 5,955,329, entitled “Engineering plant thioesterases for altered substrate specificity”, and U.S. Pat. No. 6,150,512, entitled “Engineering plant thioesterases and disclosure of plant thioesterases having novel substrate specificity”, disclose variants of plant thioesterase genes having altered chain length specificities for the encoded thioesterase enzymes.
Journal articles disclosing Class II chain acyl-ACP thioesterases include Dehesh, K. et al., “Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana”, The Plant Journal 9:167-172 (1996), Dehesh, K. et al., “Two novel thioesterases are key determinants of the bimodal distribution of acyl chain length of Cuphea palustris seed oil”, Plant Physiology 110:203-210 (1996), Dehesh, K., et al., “KAS IV: a 3-ketoacyl-ACP synthase from Cuphea sp. is a medium chain specific condensing enzyme”, The Plant Journal 15:383-390 (1998), Dörmann, P. et al., “Characterization of two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for medium-chain and oleoyl-acyl carrier protein”, Planta 189:425-432 (1993), Filichkin, S., et al., “New FATB thioesterases from a high-laurate Cuphea species: Functional and complementation analyses”, European Journal of Lipid Science and Technology 108:979-990 (2006), Slabaugh, M., et al., “Condensing enzymes from Cuphea wrightii associated with medium chain fatty acid biosynthesis”, The Plant Journal 13:611-620 (1998), Voelker, T., et al., “Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants”, Science 257:72-74 (1992), Voelker, T., and Davies, M., “Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase”, Journal of Bacteriology 176:7320-7327 (1994).
In addition to synthesizing fatty acids for nonfuel products, microorganisms or photosynthetic organisms can be used to produce fatty acids or fatty acid products for the production of fuels and chemicals such as alcohols or hydrocarbons. In synthesizing fatty acids, these organisms can use atmospheric CO2 or plant products such as starch, sugars, or cellulose that are themselves based on fixed atmospheric CO2 as a source of carbon, thereby reducing the net amount of CO2 generated in the production and use of the fuel or chemical. Increasing the yield and recovery of fatty acids and fatty acid products of a particular chain length from cultured microorganisms and photosynthetic organisms can improve the cost effectiveness of providing a renewable source of a variety of products, including fuel products.